Assessing the Therapeutic Efficacy of Proton Transport Inhibitors in a Triple-Negative Breast Cancer Murine Model with Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer Tumor pH Imaging.

Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although few methods can accurately assess their effect in vivo. In this study, we investigated whether MRI-CEST (Magnetic Resonance Imaging-Chemical Exchange Saturation Transfer) tumor pH imaging can be a useful tool to evaluate in vivo the therapeutic efficacy of several Proton Pump Inhibitors (PPIs) in breast cancer. Cell viability and extracellular pH assays were carried out in breast cancer cells cultured at physiological pH (7.4) or acid-adapted (pH of 6.5 and 6.8) following the exposure to inhibitors of V-ATPase (Lansoprazole, Esomeprazole) or NHE1 (Amiloride, Cariporide) at several concentrations. Next, triple-negative breast cancer 4T1 tumor-bearing mice were treated with Lansoprazole or Amiloride and MRI-CEST tumor pH imaging was utilized to assess the in vivo efficacy. Only Lansoprazole induced, in addition to breast cancer cell toxicity, a significant inhibition of proton extrusion. A significant reduction in tumor volume, prolonged survival, and increase in extracellular tumor pH after 1 and 2 weeks were observed after Lansoprazole treatment, whereas no significant changes were detected upon Amiloride treatment. Our results suggested that MRI-CEST tumor pH imaging can monitor the therapeutic efficacy of PPIs in breast cancer murine models.

Tumor pH Imaging Using Chemical Exchange Saturation Transfer (CEST)-MRI.

Magnetic resonance imaging (MRI) is a noninvasive imaging technique that allows for physiological and functional studies of the tumor microenvironment. Within MRI, the emerging field of chemical exchange saturation transfer (CEST) has been largely exploited for assessing a salient feature of all solid tumors, extracellular acidosis. Iopamidol-based tumor pH imaging has been demonstrated to provide accurate and high spatial resolution extracellular tumor pH maps to elucidate tumor aggressiveness and for assessing response to therapy, with a high potential for clinical translation. Here, we describe the overall setup and steps for measuring tumor extracellular pH of tumor models in mice by exploiting MRI-CEST pH imaging with a preclinical MRI scanner following the administration of iopamidol. We address issues of pH calibration curve setup, animal handling, pH-responsive contrast agent injection, acquisition protocol, and image processing for accurate quantification and visualization of tumor acidosis.

In Vivo MRI-CEST Tumor pH Imaging Detects Resistance to Proton Pump Inhibitors in Human Prostate Cancer Murine Models.

The tumor microenvironment acidification confers treatment resistance; therefore, the interference with pH regulating systems is considered a new therapeutic strategy. In this study, two human prostate cancer cell lines, PC3 and LNCaP, have been treated in vitro with proton pump inhibitors (PPIs), namely Lansoprazole, Esomeprazole (V-ATPases-inhibitors), Cariporide, and Amiloride (NHE1-inhibitors). The cell viability and pH were assessed at several drug concentrations either at normoxic or hypoxic conditions. Since Esomeprazole showed the highest toxicity towards the PC3 cancer cells compared to LNCaP ones, athymic nude mice bearing subcutaneous or orthotopic PC3 tumors were treated with Esomeprazole (dose: 2.5 mg/kg body weight) for a period of three weeks-and tumor growth was monitored. MRI-CEST tumor pH imaging with Iopamidol was performed upon treatment at 3 h, 1 week (in combination with FDG-PET), and after 2 weeks for evaluating acute, early, and late responses. Although acute tumor pH changes were observed in vivo, long-term studies on both PC3 prostate cancer models did not provide any significant change in tumor acidosis or tumor growth. In conclusion, this work shows that MRI-CEST tumor pH imaging is a valuable tool for assessing the in vivo treatment response to PPIs.

Synthetic Lethality Screening Highlights Colorectal Cancer Vulnerability to Concomitant Blockade of NEDD8 and EGFR Pathways.

Colorectal cancer (CRC) is a heterogeneous disease showing significant variability in clinical aggressiveness. Primary and acquired resistance limits the efficacy of available treatments, and identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a "drop-out" loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. These results unveil possible therapeutic opportunities in specific CRC clinical settings.

Extrafollicular IgD+ B cells generate IgE antibody secreting cells in the nasal mucosa.

Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naïve-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naïve B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

Non-invasive Investigation of Tumor Metabolism and Acidosis by MRI-CEST Imaging.

Altered metabolism is considered a core hallmark of cancer. By monitoring in vivo metabolites changes or characterizing the tumor microenvironment, non-invasive imaging approaches play a fundamental role in elucidating several aspects of tumor biology. Within the magnetic resonance imaging (MRI) modality, the chemical exchange saturation transfer (CEST) approach has emerged as a new technique that provides high spatial resolution and sensitivity for in vivo imaging of tumor metabolism and acidosis. This mini-review describes CEST-based methods to non-invasively investigate tumor metabolism and important metabolites involved, such as glucose and lactate, as well as measurement of tumor acidosis. Approaches that have been exploited to assess response to anticancer therapies will also be reported for each specific technique.

Endocannabinoid receptor CB2R is significantly expressed in aspirin-exacerbated respiratory disease: a pilot study.

BACKGROUND: The endocannabinoid system represents a highly conserved, innate signaling network with direct and indirect control of eicosanoid-mediated inflammation. Activation of the type 2 cannabinoid receptor (CB2R) leads to decreased type 2 inflammation and reduced production of arachidonic acid (AA). Given that altered AA metabolism is associated with aspirin-exacerbated respiratory disease (AERD), we hypothesized that expression of the CB2R gene CNR2 is increased in AERD. METHODS: Nasal polyps from consecutive patients undergoing endoscopic sinus surgery for AERD or allergic fungal rhinosinusitis (AFRS) were prospectively evaluated. Control sphenoid mucosa was collected from patients undergoing endoscopic skull base procedures. Expression and localization of endocannabinoid receptors were evaluated by quantitative reverse transcript-polymerase chain reaction (qRT-PCR) and immunohistochemistry. A 2-group unpaired t test with unequal variances was used to evaluate group differences. RESULTS: Thirteen subjects were included in this pilot study, including 5 controls, 5 AFRS patients, and 3 AERD patients. Upregulated expression of CNR2 was detected in subjects with AERD vs both AFRS (p = 0.049) and controls (p = 0.047), with a mean increase of 5.2-fold. No significant differences in expression of the CB1R gene CNR1 were detected between control and AFRS groups. Immunohistochemistry predominantly localized CB1R and CB2R expression to the surface epithelium in all subjects. CONCLUSION: The endocannabinoid system is an emerging immunomodulatory network that may be involved in AERD. This is the first study of CB2R in sinonasal disease, showing significantly increased transcription in nasal polyps from subjects with AERD. Additional study is warranted to further evaluate the contribution and therapeutic potential of this novel finding in chronic rhinosinusitis.

TAZ/WWTR1 is overexpressed in papillary thyroid carcinoma.

In this study, we analysed the expression of the transcriptional coactivator TAZ (transcriptional co-activator with PDZ-binding motif), also named WWTR1, in a panel of papillary thyroid carcinoma samples and we observed a significant deregulation of its expression in such tumours. Specifically, by quantitative real-time PCR (qRT-PCR) we evaluated TAZ mRNA levels in tissue specimens (n=61) of papillary thyroid carcinoma (PTC) and herein we show that the PTC samples express much higher TAZ mRNA levels with respect to the normal thyroid tissue (p<0.001). TAZ expression was also evaluated in normal (n=10) and pathological human thyroids (n=17) by immunohistochemical analysis and the increase of TAZ protein levels in PTC was confirmed. To further analyse the molecular mechanisms underlying TAZ overexpression in PTC, we used an inducible system consisting of FRTL-5 rat thyroid cells expressing a conditional RAS oncoprotein and we show that the activation of the RAS signalling pathway is involved in TAZ deregulation. These observations suggest that the activated effectors of the RAS/RAF/MEK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) signalling pathway are involved in the increased expression of TAZ, supporting the idea that this may also occur in thyroid papillary carcinoma. Moreover, we demonstrated that the overexpression of TAZ is able to confer growth advantage to thyroid cells in culture and to induce epithelial-mesenchymal transition. In conclusion, these findings support a potential role for TAZ in the pathogenesis of papillary thyroid carcinomas.